This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Development of metal ion affinity chromatography (IMAC) and titanium dioxide (TiO2) based methodology for the isolation of phospho-peptides at the sub-picomole level. These enrichment techniques will be combined with off-line multidimensional chromatography fractionation prior to final analysis using reverse phase LC-MS/MS. Applications to determination of sites of phosphorylation on a variety of biologically important proteins, either in an isolated state or in the context of complex mixtures. (Additional effort and instrument time reported under Collaborative projects and other Technical Research and Development project.)